EC number 3.2.1.4 IntEnz IntEnz view ExPASy NiceZyme view | CAS number 9012-54-8 BRENDA BRENDA entry KEGG KEGG entry | |
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Cellulase is any of several enzymes produced chiefly by fungi, bacteria, and protozoans that catalyze cellulolysis, the decomposition of cellulose and of some related polysaccharides. The name is also used for any naturally occurring mixture or complex of various such enzymes, that act serially or synergistically to decompose cellulosic material.
Contents
- Types and action
- Structure
- Cellulase complexes
- Uses
- Measurement of cellulase
- Viscometric substrates
- Cellooligosaccharide substrates
- Reduced cellooligosaccharide substrates
- Dyed polysaccharide substrates
- Enzyme coupled reagents
- References
Cellulases break down the cellulose molecule into monosaccharides ("simple sugars") such as beta-glucose, or shorter polysaccharides and oligosaccharides. Cellulose breakdown is of considerable economic importance, because it makes a major constituent of plants available for consumption and use in chemical reactions. The specific reaction involved is the hydrolysis of the 1,4-beta-D-glycosidic linkages in cellulose, hemicellulose, lichenin, and cereal beta-D-glucans. Because cellulose molecules bind strongly to each other, cellulolysis is relatively difficult compared to the breakdown of other polysaccharides such as starch.
Most mammals have only very limited ability to digest dietary fibres such as cellulose by themselves. In many herbivorous animals such as ruminants like cattle and sheep and hindgut fermenters like horses, cellulases are produced by symbiotic bacteria. Cellulases are produced by a few types of animals, such as some termites.
Several different kinds of cellulases are known, which differ structurally and mechanistically. Synonyms, derivatives, and specific enzymes associated with the name "cellulase" include endo-1,4-beta-D-glucanase (beta-1,4-glucanase, beta-1,4-endoglucan hydrolase, endoglucanase D, 1,4-(1,3,1,4)-beta-D-glucan 4-glucanohydrolase), carboxymethyl cellulase (CMCase), avicelase, celludextrinase, cellulase A, cellulosin AP, alkali cellulase, cellulase A 3, 9.5 cellulase, and pancellase SS. Enzymes that cleave lignin are occasionally called cellulases, but this is usually considered erroneous.
Types and action
Five general types of cellulases based on the type of reaction catalyzed:
Avicelase has almost exclusively exo-cellulase activity, since avicel is a highly micro-crystalline substrate.
Within the above types there are also progressive (also known as processive) and nonprogressive types. Progressive cellulase will continue to interact with a single polysaccharide strand, nonprogressive cellulase will interact once then disengage and engage another polysaccharide strand.
Cellulase action is considered to be synergistic as all three classes of cellulase can yield much more sugar than the addition of all three separately. Aside from ruminants, most animals (including humans) do not produce cellulase in their bodies and can only partially break down cellulose through fermentation, limiting their ability to use energy in fibrous plant material.
Structure
Most fungal cellulases have a two-domain structure, with one catalytic domain and one cellulose binding domain, that are connected by a flexible linker. This structure is adapted for working on an insoluble substrate, and it allows the enzyme to diffuse two-dimensionally on a surface in a caterpillar-like fashion. However, there are also cellulases (mostly endoglucanases) that lack cellulose binding domains. These enzymes might have a swelling function.
Cellulase complexes
In many bacteria, cellulases in-vivo are complex enzyme structures organized in supramolecular complexes, the cellulosomes. They contain roughly five different enzymatic subunits representing namely endocellulases, exocellulases, cellobiases, oxidative cellulases and cellulose phosphorylases wherein only endocellulases and cellobiases participate in the actual hydrolysis of the β(1→ 4) linkage.
The cellulase complex from Trichoderma reesei, for example, comprises a component labeled C1 (57,000 daltons) that separates the chains of crystalline cellulose, an endoglucanase (about 52,000 daltons), an exoglucanase (about 61,000 dalton), and a beta-glucosidase (76,000 daltons).
Numerous "signature" sequences known as dockerins and cohesins have been identified in the genomes of bacteria that produce cellulosomes. Depending on their amino acid sequence and tertiary structures, cellulases are divided into clans and families.
Uses
Cellulase is used for commercial food processing in coffee. It performs hydrolysis of cellulose during drying of beans. Furthermore, cellulases are widely used in textile industry and in laundry detergents. They have also been used in the pulp and paper industry for various purposes, and they are even used for pharmaceutical applications. Cellulase is used in the fermentation of biomass into biofuels, although this process is relatively experimental at present. Cellulase is used as a treatment for phytobezoars, a form of cellulose bezoar found in the human stomach.
Measurement of cellulase
As the native substrate, cellulose, is a water-insoluble polymer, traditional reducing sugar assays using this substrate can not be employed for the measurement of cellulase activity. Analytical scientists have developed a number of alternative methods.
Viscometric substrates
A viscometer can be used to measure the decrease in viscosity of a solution containing a water-soluble cellulose derivative such as carboxymethyl cellulose upon incubation with a cellulase sample. The decrease in viscosity is directly proportional to the cellulase activity. While such assays are very sensitive and specific for endo-cellulase (exo-acting cellulase enzymes produce little or no change in viscosity), they are limited by the fact that it is hard to define activity in conventional enzyme units (micromoles of substrate hydrolyzed or product produced per minute).
Cellooligosaccharide substrates
The lower DP cello-oligosaccharides (DP2-6) are sufficiently soluble in water to act as viable substrates for cellulase enzymes. However, as these substrates are themselves 'reducing sugars', they are not suitable for use in traditional reducing sugar assays because they generate a high 'blank' value. However their cellulase mediated hydrolysis can be monitored by HPLC or IC methods to gain valuable information on the substrate requirements of a particular cellulase enzyme.
Reduced cellooligosaccharide substrates
Cello-oligosaccharides can be chemically reduced through the action of sodium borohydride to produce their corresponding sugar alcohols. These compounds do not react in reducing sugar assays but their hydrolysis products do. This makes borohydride reduced cello-oligosaccharides valuable substrates for the assay of cellulase using traditional reducing sugar assays such as the Nelson-Symogyi method.
Dyed polysaccharide substrates
These substrates can be subdivided into two classes-
Enzyme coupled reagents
Recently, new reagents have been developed that allow for the specific measurement of endo-cellulase. These methods involve the use of functionalised oligosaccharide substrates in the presence of an ancillary enzyme. In the example shown, a cellulase enzyme is able to recognise the trisaccharide fragment of cellulose and cleave this unit. The ancillary enzyme present in the reagent mixture (β-glucosidase) then acts to hydrolyse the fragment containing the chromophore or fluorophore. The assay is terminated by the addition of a basic solution that stops the enzymatic reaction and deprotonates the liberated phenolic compound to produce the phenolate species. The cellulase activity of a given sample is directly proportional to the quantity of phenolate liberated which can be measured using a spectrophotometer. The acetal functionalisation on the non-reducing end of the trisaccharide substrate prevents the action of the ancillary β-glucosidase on the parent substrate.